Project Summary/Abstract: Chronic pain is a significant health problem in the United States and in which there are limited treatment options available. Opioid drugs are commonly used in the treatment of chronic pain but carry with them serious side effects such as hyperalgesia (increased sensitivity to painful stimuli) and tolerance. These two side effects limit the effectiveness of opioids pain relieving ability and make the patient more susceptible to overdose. Previous research has shown opioids alter the gut microbiota. Downstream effects of an altered microbiota include increased gut inflammation, bile acid dysregulation, and change in vagal nerve and cortical activity. Preliminary data show that chronic opioid use alters short-chain fatty acid (SCFA) production in the gut and that modulating the gut microbiome through the ketogenic diet inhibits the maintenance of opioid induced hyperalgesia and tolerance. The direct link from the gut to the brain is through the vagal nerve. Recent work has shown that vagal nerve stimulation can modulate neuronal activity in the limbic system, including anterior cingulate cortex (ACC), a critical brain structure to be involved in chronic pain processing. Taken together, these findings lead to a central hypothesis that chronic opioid use causes microbiota changes in the gut which directly contributes to opioid induced hyperalgesia and tolerance by altering neuronal activity in the ACC through the vagal nerve, and that peripherally (ketogenic diet) or centrally (neuromodulation) targeted treatment modalities may be used to treat the opioid induced hyperalgesia and tolerance. Two aims are proposed to test this central hypothesis and to demonstrate that modulation of gut-brain axis by a dietary or neuromodulation approach can serve as potential therapeutic options. Aim 1: Establish the role of gut microbiota in the therapeutic effect of a special diet (ketogenic diet) on fentanyl induced hyperalgesia and tolerance under chronic pain by a) analyzing microbiota changes b) altering the microbiota with fecal microbiota transplantation or antibiotic administration c) measuring SCFAs and bile acids to detect the functional consequences of an altered microbiota due to chronic fentanyl treatment. Aim 2: Determine the effect of altered neuronal activity on fentanyl induced hyperalgesia and tolerance under chronic pain by a) measuring vagal nerve activity during chronic pain and opioid treatment b) examining the effect of altering vagal nerve activity on fentanyl induced hyperalgesia and tolerance c) investigating whether modulating ACC neuronal activity can treat fentanyl induced hyperalgesia and tolerance. Completion of this proposed project will provide new insight into how altered gut-brain signaling can lead to behavioral changes and how modulation of gut-brain axis can be used as a treatment strategy for fentanyl induced hyperalgesia and tolerance.